3T3 fibroblasts release a low-molecular-weight inhibitory peptide of lymphocyte DNA synthesis into serum-free culture medium

3T3 fibroblasts release a novel factor into serum-free culture medium, which strongly suppressed concanavalin A-induced thymocyte DNA synthesis. This activity was highly purified by gel filtration, ion exchange and thin-layer chromatography and was characterized as a 1 kDa heat-stable peptide. Although this peptide suppressed lymphocyte DNA synthesis when added relatively early after lectin-stimulation, the cell viability was not changed significantly. The peptide considerably repressed DNA synthesis of some mammalian fibroblast cells, but malignant-transformed cells were not affected.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

A uterine cell mitogen distinct from epidermal growth factor in porcine uterine luminal fluids: characterization and partial purification

Uterine luminal fluids (ULF) from early (Days 10 and 12)-pregnant sows contain factors that stimulate DNA synthesis in a variety of cell lines. The major growth factor component in these fluids has been partially purified 200-fold by heat treatment, anion-exchange chromatography, and gel filtration using mouse embryo-derived AKR-2B fibroblasts as an indicator cell line. The ULF mitogen (ULFM) is a polypeptide with an apparent molecular weight of 4800; it is extremely heat stable and resistant to treatment with urea. This mitogen is also present in ULF from cycling sows but is not detectable in uterine cytosolic extracts or in serum isolated from pigs at Day 12 of pregnancy. The addition of this factor to medium containing 0.5% calf serum results in a 50% increase in final cell density of AKR-2B cells. ULFM appears biologically distinct from mouse and human epidermal growth factor (EGF), since its activity is not inhibited by antibody to mouse EGF and it does not compete for binding to human (A431) EGF receptors. In addition, the ULF factor stimulates DNA synthesis in human A431 epidermoid carcinoma cells, whereas EGF is inhibitory. Partially purified ULFM also stimulates DNA synthesis in primary cultures of pig uterine stromal cells. This mitogen activity is dose-dependent and is not inhibited by antibody to mouse EGF. Thus ULFM may act in concert with other peptide growth factors in regulating uterine growth and/or differentiation.     

Regulation of growth of human diploid fibrobalasts by factors elaborated by activated lymphoid cells

Fibroblast growth and synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80°C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (α-LT). It was not cytotoxic, as shown by the ⁵¹chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G⁰ or G¹ stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA Synthesis but cannot divide, and they accumulate in G². The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.     

Synthesis of sterol and phospholipid induced by the interaction of phytohemagglutinin and other mitogens with human lymphocytes and their relation to blastogenesis and DNA synthesis

Human peripheral blood lymphocytes (PBL) responded to phytohemagglutinin (PHA) and a variety of other mitogens by increased synthesis of sterol and phospholipid. This activity was established within 4–7 hr of the addition of mitogen and was dependent upon the binding of the ligand to the cell membrane. Sterol and phospholipid synthesis reached a peak at approximately 24 hr in association with blastogenic expansion of the lymphocyte membrane and initiation of DNA synthesis. Lipid synthesis and blast transformation occurred independently of replication of the genome since inhibition of DNA synthesis did not reduce the degree of blast transformation and lipid synthesis observed. However, inhibition of sterol synthesis using 20α-hydroxycholesterol resulted in decreased blastogenesis and DNA synthesis, demonstrating that early synthesis of lipid is important for these subsequent events. Human thymocytes responded to T-cell mitogens in the conventional manner as regards synthesis of lipid and blast transformation; however, they did not synthesize DNA. Possible reasons for this incomplete response are discussed. Several nonmitogenic agents which agglutinate lymphocytes were also found to initiate early increases in sterol and phospholipid synthesis, and the possible significance of this observation is considered.     

Deoxyadenosine (AdR) inhibition of newly activated lymphocytes: blockade at the G0-G1 interface

The mechanism responsible for the lymphocytotoxicity associated with congenital adenosine deaminase (ADA) deficiency has been ascribed to an accumulation of dATP. Elevated levels of dATP can then lead to inhibition of DNA synthesis by inhibiting ribonucleotide reductase and causing a depletion of the other deoxynucleotide triphosphates (dNTP). This hypothesis was derived principally from studies with murine and human lymphoblastoid cell lines (LCL) and apparently confirmed in a limited number of investigations with lectin-stimulated lymphocytes. Our biochemical studies of lectin-stimulated mouse and human lymphocytes were not consistent with the dATP model and suggested that AdR exerted effects on lymphocyte activation that preceded the initiation of DNA synthesis. In the current studies, we focused on the effects of AdR on the early events in T lymphocyte activation, because we found they were the most sensitive to AdR toxicity. AdR blocked neither the production of T cell growth factor (TCGF) by lectin-stimulated lymphocytes nor the expression of TCGF receptors as detected by the anti-Tac monoclonal antibody that recognizes the human TCGF receptor. AdR did, however, block the early TCGF-dependent events leading to the entry into the cell cycle. By using the metachromatic fluorescence stain acridine orange, we found that AdR blocked the increased synthesis of RNA that characterizes the entry into the G1 phase of the cell cycle from the G0, resting state. Because these early effects were caused by the lowest doses of AdR, and because they preceded the synthesis of DNA by 15 to 20 hr, it suggested that these effects may be principally responsible for the in vivo toxicity associated with ADA deficiency. Furthermore, none of the other proposed biochemical mechanisms, e.g., inhibition of methylation, diminution of ATP levels, or incorporation of AdR into polyadenylated RNA, appeared adequate to explain AdR toxicity during T lymphocyte activation.     

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D₁ (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-I (lymphocyte activating factor).     

Further characterization of the phenotype of ts20, a DNAts mutant of BALB/3T3 cells

Ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells that is blocked at a step in DNA synthesis involving chain elongation. Following a shift from 33 degrees to 39 degrees C, mutant cells lost ability to grow or form colonies. When mutant cells were infected with polyomavirus, both cell and virus DNA synthesis were inhibited at the restrictive temperature of 39 degrees C. When cell extracts from wild-type cells were added in vitro to lysed infected mutant cells that had been incubated in vivo at 39 degrees C for expression of the mutation, cell DNA synthesis was increased 3-fold (similar to the effect in uninfected mutant cells), whereas virus DNA synthesis was increased only 60%. With harsher lysis conditions, the effect of added extract on virus DNA synthesis was greater, although baseline DNA synthesis (prior to addition of extracts) was much lower. Analysis by alkaline sucrose gradients showed that the addition of cell extract converted small cellular DNA molecules into larger ones, while it increased the synthesis of small virus DNA molecules rather than completed genomes. Analysis of cytosol extracts (in which the activity stimulating DNA synthesis resides) showed that DNA topo-isomerase I activity was more heat-labile when assayed in mutant extracts compared to wild-type extracts. In contrast, cytosol DNA polymerase activity was equally heat-labile in mutant and wild-type extract. This suggested the factor in extract was likely associated with the activity of DNA topo-isomerase I. Analysis of virus DNA synthesized in vitro in restricted mutant cells by gel electrophoresis and fluorography showed an accumulation of topo-isomers migrating between form I and II. These topo-isomers, thought to be a manifestation of the ts defect, did not disappear when extract from wild-type cells was added back in vitro or when mutant cells were shifted back to permissive temperature prior to lysis for in vitro synthesis. The results indicate that polyoma DNA synthesis and cell DNA synthesis differ in their response to the mutant gene product in ts20, although both are inhibited at a step early in DNA chain elongation that may involve DNA topo-isomerase I.     

Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.     

Mechanism of hepatic ornithine decarboxylase induction by the ornithine decarboxylase-inducing factor isolated from tumor ascites fluid: determination of target cells for the factor in the liver

The effect of the ODC-factor, which was partially purified from ascites fluid of mice bearing Ehrlich ascites hepatoma, on DNA synthesis in the normal mouse liver and spleen was studied, and target cells for the factor in the liver were examined. DNA synthesis in the liver increased about 4-fold over the basal level 39-42 h after the increase of ODC activity induced by injection of the factor into normal mice. This increase of DNA synthesis was inhibited by repeated injection of DAPol. The inhibition was completely reversed by the administration of an appropriate amount of putrescine at about the same time. TK activity also increased in parallel with DNA synthesis. Normal mice with and without treatment with the factor were used to examine which cell population in the liver is the real target for the factor. The livers were dispersed and three cell populations (heavy, medium, and light) were separated by centrifugation. The heavy and light cell populations were characterized as mature hepatocytes and a cell population consisting mainly of immature hepatocytes and non-hepatocytes by analysis of marker enzymes, pyruvate kinase isozymes, L and M2, respectively. The factor stimulated ODC induction, with concomitant increases in TK and DNA poly activities and DNA synthesis, most effectively in the light cell fraction followed in order by the medium and heavy fractions. A nutritional factor (a high protein diet), which is a potent inducer of liver ODC, appeared to act on liver in a different way from the ODC-factor, judging from the results of studies of both whole liver and the fractionated cell system described above. Autoradiography of [3H]thymidine incorporation into liver cells showed that DNA synthesis in mature hepatocytes as well as nonhepatocytes was enhanced by injection of the factor. Stimulation of non-hepatocytes seems to be suggestive evidence that an immunologic response of mice might be developed by the factor. In fact, ODC activity, DNA synthesis, and DNA poly activity (but not TK) in the spleen significantly increased in response to the factor and their increments were suppressed by DAPol, though less sensitively than those in the light cell fraction of the liver.     

Purification of primate T cell growth factor to apparent homogeneity by reverse phase high pressure liquid chromatography: evidence for two highly active molecularly distinct species

T cell growth factor produced by the MLA144 gibbon ape lymphosarcoma T lymphocyte line was separated into two molecular forms of Mr 16,300 and Mr 14,300 which were purified 32,000-fold and 82,000-fold, respectively, and which in combination constitute the majority of biological activity of the starting material. The overall recovery of biological activity was 50%. This was accomplished with a series of chromatographic steps including reverse-phase high pressure liquid chromatography. The purified proteins are of comparable specific activities and each maintains the DNA synthesis of T cell growth factor-dependent human T cell lines at concentrations of less than approximately 18 pM (300 fg/ml).     

A permeable FGF-1 nuclear localization sequence peptide induces DNA synthesis independently of Ras activation

A 26-amino-acid peptide (designated PFNP) composed of the nuclear localization signal of fibroblast growth factor (FGF)-1 and a membrane-permeable peptide is known to mimic FGF-1's ability to stimulate DNA synthesis in various cell types at low cell densities. The underlying molecular mechanism is unknown, however. Here we show that PFNP activity is inhibited in murine fibroblasts by a tyrosine kinase inhibitor, that PFNP does not bind to the FGF receptor, and that PFNP does not induce phosphorylation of the FGF receptor substrate. In addition, expression of a dominant-negative form of Ras, which abolished the activities of epidermal growth factor (EGF) and heparin-binding EGF, had no affect on PFNP-induced DNA synthesis. Despite this apparent Ras independence, PFNP activity correlated with phosphorylation of ERK1/2 MAP kinases and was concentration dependently inhibited by inhibitors of ERK1/2 MAP kinase phosphorylation. These results indicate that whereas Ras activation is dispensable for PFNP-induced DNA synthesis, activation of tyrosine kinases and ERK1/2 kinases, albeit independently of the FGF receptor system, is crucial. Interestingly, FGF-1 signaling was predominantly Ras-independent when the cell density was optimum for PFNP, suggesting that PFNP and FGF-1 share the same signaling mechanism.     

Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10(-4) M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56 degrees C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10(-4) M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10(-4) M spermidine.     

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.     

Inhibition of the mixed lymphocyte proliferative response by phorbol esters.

The phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, a potent cocarcinogen in mice, blocks the induction of DNA synthesis in lymphocytes undergoing the mixed lymphocyte response. At 10(-7) M diester, the induced DNA synthesis is inhibited almost completely (99%). This action of the diester affects some early step in the response which is necessary for the triggering of cell replication; on-going DNA replication is not significantly affected. Phorbol 12,13-diacetate, a less potent analogue in tumor promotion in vivo, is also a less potent inhibitor of the mixed lymphocyte response (75% inhibition at 10(-6) M). Phorbol, the parent alcohol, is not effective in either system. The use of phorbol diesters in the molecular dissection of mixed lymphocyte responses is discussed.     

Isolation and characterization of a siderophore-like growth factor from mutants of SV40-transformed cells adapted to picolinic acid.

We have isolated mutants of SV40-transformed BALB/3T3 cells adapted to grow in picolinic acid. A line of cells, derived from the mutant cells, that multiplies in the absence of serum has also been characterized. From ultrafiltrates of medium conditioned by contact with these cell lines, we have identified and partially purified a highly specific iron-binding ligand termed siderophore-like growth factor (SGF). Experiments have indicated that the factor is a peptide(s) of approximately 1600 daltons. In nanogram amounts, SGF solubilizes and binds Fe 3+ in vitro and stimulates the uptake of Fe 3+ in vivo. The Fe 3+ uptake preceded the stimulation of DNA synthesis induced by the factor in the mutant cell lines. The factor could not be separated into Fe 3+ binding and DNA synthetic activity, suggesting that the early iron uptake is an integral component of the proliferative response. These data support the hypothesis that SGF is representative of a family of mammalian siderophores which may have an important role in cell proliferation.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

Summary The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10⁻⁴ M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56°C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10⁻⁴ M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10⁻⁴ M spermidine. This work was supported in part by the Cystic Fibrosis Foundation.     

Isolation of an autocrine growth factor from hepatoma HTC-SR cells

A growth factor has been isolated from HTC-SR rat hepatoma tissue culture cells which specifically stimulates DNA synthesis and cell proliferation of the HTC cells that produce it. The factor can be isolated from HTC cell conditioned medium or from an HTC cell extract. This autocrine factor has been purified 640-fold from a postmicrosomal supernatant by successive steps, involving ethanol precipitation, heating at 80 degrees C for 10 min, chromatography on a DEAE Bio-Gel A column, and chromatography on a heparin-sepharose affinity column. The major peak of activity eluted from the heparin column migrates as a single band on SDS-PAGE with an apparent Mr of 60,000. The factor is resistant to acid, heat, and neuraminidase but sensitive to trypsin, papain, and protease. The autocrine nature of the factor is indicated by the finding that several other types of cells do not respond with increased DNA synthesis. Mouse L-cells, BHK cells, Novikoff hepatoma cells, hepatocytes in primary culture, and an epithelial-like rat liver-derived cell line (Clone 9) were tested, and none of the cells could be stimulated. Small amounts of the factor could be extracted from the Clone 9 cells, however. This material had the same physical and purification properties as the factor extracted from HTC cells, but it did not stimulate DNA synthesis in Clone 9 cells, only in HTC cells. Addition of the factor resulted in an almost immediate stimulation of DNA synthesis in a proliferating HTC cell population. When the factor was added together with [3H]thymidine for 2 h, a significant stimulation of DNA synthesis was observed, provided the addition was made between 18 and 48 h after the cells had been plated. Autoradiographic studies indicated that the factor both accelerates DNA synthesis in cells already making DNA and increases the number of cells entering the S period. The stimulation of DNA synthesis was completely inhibited by 10 mM hydroxyurea, whether the factor was present for 2, 24, or 48 h in the culture. A significant increase in cell number due to addition of the factor was also observed. This accelerated proliferation was detectable only after the cells had been in culture for at least 48 h with the factor present.